With the emergence of bacterial resistance, identification of new diseases, and the need for new therapeutics with different efficacies, our ability to design drugs to battle bacterial infections is becoming a more urgent priority. Natural products are often useful as therapeutics for humans, though problems such as side effects and production difficulties can preclude their successful development. This proposal seeks to address the need for new antibiotics by studying structure-function relationships in the valanimycin biosynthetic pathway. This naturally available antibiotic has efficacy against gram positive and gram negative bacteria, and shows some promise as an anticancer therapeutic. With this research we hope to make valanimycin amenable to a new drug development strategy, synthetic biology, in which the drug's biosynthetic pathway is engineered to allow introduction of diversity into the product. The research described here focuses on a biosynthetic step common to multiple antibiotics - flavin-dependent hydroxylation of a primary amine. The enzyme responsible for this step in the valanimycin biosynthetic pathway will be structurally and biochemically characterized. The structure activity relationships identified by those studies will be verified by bioinformatics and biochemical techniques, including mutagenesis combined with enzymatic activity and binding studies. The data yielded will enable rational design of vlmH to alter its substrate binding specificity. Such studies can be pursued on other steps of the pathway; in this way, we can introduce diversity into the valanimycin final structure. Given enough time and research, this molecule could be developed into a useful therapeutic.